Method for preparing liquid medium composition, and preparation device and kit therefor

ABSTRACT

The present invention provides a method capable of easily mixing any liquid containing a linking substance such as a divalent metal cation and the like with a liquid containing a particular compound at a high concentration, and capable of producing a liquid medium composition comprising fine structures dispersed therein, and a production device therefor and a kit therefor. The first liquid containing a particular compound is passed through a through-hole having a given cross-sectional area formed in a nozzle part at a given flow rate and injected into the second liquid at a given flow rate. By this simple operation, a structure in which the particular compound is bonded via the linking substance is formed, and the structure is preferably dispersed in a mixture of the both liquids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of copending U.S. patentapplication Ser. No. 15/565,022, filed on Oct. 6, 2017, which is theU.S. national phase of International Patent Application No.PCT/JP2016/061357, filed on Apr. 7, 2016, which claims the benefit ofJapanese Patent Application No. 2015-078795, filed on Apr. 7, 2015, thedisclosures of which are incorporated herein by reference in theirentireties for all purposes.

TECHNICAL FIELD

The present invention relates to a production method of a liquid mediumcomposition, and a production device and a kit for carrying out themethod. More particularly, the present invention relates to a productionmethod including appropriately mixing at least two kinds of liquids tobe mixed for forming the above-mentioned medium composition (the firstliquid containing a particular compound, and the second liquidcontaining a substance for linking the particular compounds to form astructure) to produce a liquid medium composition comprising theabove-mentioned structure dispersed therein, a production device and akit enabling the dispersing thereof.

BACKGROUND ART

In recent years, techniques for proliferating or maintaining in vitrovarious organs, tissues and cells that play distinct roles in the bodyof animals and plants have been developed. Proliferation or maintenanceof the organs and tissues in vitro is called organ culture and tissueculture, respectively, and proliferating, differentiating or maintainingin vitro the cells separated from an organ or tissue is called cellculture.

Cell culture is a technique for proliferating, differentiating ormaintaining separated cells in vitro in a medium, and is indispensablefor detailed analyses of the in vivo function and structure of variousorgans, tissues and cells. In addition, the cells and/or tissuescultured by the technique are utilized in various fields includingefficacy and toxicity evaluation of chemical substances, pharmaceuticalproducts and the like, large-scale production of useful substances suchas enzymes, cell growth factors, antibodies and the like, regenerativemedicine supplementing organ, tissue and cells that were lost by diseaseand deficiency, improvement of plant brand, production of generecombinant products, and the like.

As a medium for culturing cells and the like (organ, tissue, cells), aliquid medium can be mentioned, and the present inventors successfullydeveloped a liquid medium composition enabling culture of cells and thelike in a suspending state (patent documents 1 and 2).

The liquid medium composition described in patent document 1 is one inwhich particular compounds (particularly, a polymer compound having ananionic functional group) assemble via a divalent metal cation and thelike to form amorphous structures, and the structures are dispersed in aliquid medium in a suspended state. In the following, theabove-mentioned particular compound such as a polymer compound having ananionic functional group and the like is also referred to as a“particular compound”, and a substance such as a divalent metal cationand the like which binds the particular compounds is also referred to asa “linking substance”.

The medium composition provides a preferable liquid medium capable ofculturing cells and the like in a suspended state without accompanyingan operation such as shaking, rotation and the like having a risk ofcausing damage and loss of functions of cells and the like.

DOCUMENT LIST Patent Documents

patent document 1: WO 2014/017513patent document 2: US 2014/0106348 A1

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The originally-intended preferable state of the liquid mediumcomposition described in the above-mentioned patent document 1 is astate in which small amounts of the particular compounds are linked toeach other to form fine structures and many structures are dispersed ina liquid.

However, the present inventors studied the actual production steps ofthe liquid medium composition in detail, and found that to achieve suchpreferable state, it is necessary to ensure that the structure is notunevenly formed locally in the medium composition. For example, when theparticular compound is deacylated gellan gum, it forms an indeterminateamorphous structure via a linking substance ((e.g., calcium ion) in aliquid medium when mixed with the liquid medium, and the structurebecomes a carrier for suspending cells and the like. However, in amixing method including pouring a liquid containing a particularcompound at a high concentration into a liquid medium containing alinking substance while stirring the medium, the particular compoundcontacts with the linking substance at the moment the both liquids flowtogether to form the structure. As a result, the structures aresometimes linked to form a long string suspended in the mixture (orstring structure entangled in a mass), which is not theoriginally-intended dispersed state. It was also found that such stateis developed even when the mixture is stirred at a comparatively highspeed. Once such string structure is formed in the liquid medium, it isnot easy to cleave the structure finely and disperse same in the basematerial in view of the property of the structure that a double helixformed by the molecular chain forms a tight 3-dimensional network witheach other via a linking substance (e.g., calcium ion).

To afford a state of the structure finely dispersed in the liquidmedium, a special treatment for dispersing may be performed, whichincludes, for example, using a powder medium or a liquid medium of knowncomponents such as concentrated medium and the like, diluting a liquidcontaining a particular compound, and mixing them to produce a state ofdispersion of fine structures.

However, such special treatment for dispersing takes time and effort andis sometimes difficult to perform on conventional liquid medium andspecial liquid medium, which in turn limits the cells and the like to becultured.

An object of the present invention is to solve the above-mentionedproblem, and provides a method capable of easily mixing any liquidcontaining a linking substance such as a divalent metal cation and thelike with a liquid containing a particular compound at a highconcentration, and capable of producing a liquid medium compositioncomprising fine structures dispersed therein, and a production devicetherefor and a kit therefor.

Means of Solving the Problems

The present inventors have conducted intensive studies and found that aliquid medium composition comprising a liquid medium and fine structurespreferably dispersed in the liquid medium can be obtained without usinga special stirring apparatus and by passing a liquid containing aparticular compound through a through-hole having a cross section in aparticular range at a flow rate not less than a particular value, andmerely injecting the liquid at said flow rate into a liquid containing alinking substance, which resulted in the completion of the presentinvention.

The main constitution of the present invention is as follows.

[1] A production method of liquid medium composition, comprising stepsof:

passing a first liquid comprising a particular compound of the following(i) through a through-hole having a cross-sectional area of 0.01mm²-5.00 mm² formed in a nozzle part at a flow rate of not less than 1.7mL/sec,

injecting the first liquid into a second liquid comprising a linkingsubstance of the following (ii) at said flow rate to form structures inwhich the particular compounds are bound via the linking substance anddisperse the structures in a mixture of the both liquids mentionedabove:

(i) a particular compound which is a polymer compound having an anionicfunctional group, and capable of forming a structure by linking via adivalent metal cation, which structure being capable of suspending acell or a tissue,

(ii) a linking substance which is a divalent metal cation.

[2] The production method of the above-mentioned [1], wherein the secondliquid is placed in a container of the following (a), the first liquidis fed out from a supply device and passed through the through-hole of acontainer of the following (a) at the above-mentioned flow rate, wherebythe first liquid is injected into the second liquid in the container atthe above-mentioned flow rate:

(a) a container comprising a body and a lid, said body or lid providedwith a nozzle part having a through-hole communicating the outside ofthe container and the inside of the container, and said through-holehaving a cross-sectional area of 0.01 mm²-5.00 mm².

[3] The production method of the above-mentioned [2], wherein the supplydevice is a syringe, and

the nozzle part is provided on the lid of the container, and a tubularcomponent for fitting a syringe tip protrudes from an outer surface ofthe lid at a container external side of the nozzle part.

[4] The production method of the above-mentioned [1], wherein the secondliquid is placed in a container of the following (a), a tip of a nozzlepart of a supply device of the following (b) containing the first liquidis inserted into the container, the first liquid is fed out from thesupply device and passed through the through-hole in the nozzle part ofthe supply device, whereby the first liquid is injected into the secondliquid in the container at the above-mentioned flow rate:

(b) a supply device comprising a container part for containing a liquid,and a nozzle part for injecting the contained liquid through athrough-hole, said through-hole having a cross-sectional area of 0.01mm²-5.00 mm².

[5] The production method of the above-mentioned [4], wherein the supplydevice is a syringe, and the nozzle part is an injection needle mountedon the syringe,

the lid of the container is provided with a penetratable part permittingpenetration of a needle tube part of the injection needle from theoutside of the container to the inside of the container, and a tubularcomponent for fitting a needle base part of the injection needleprotrudes from an outer surface of the lid at a container external sideof the penetratable part.

[6] The production method of any one of the above-mentioned [1] to [5],wherein

the particular compound of (i) is deacylated gellan gum, the firstliquid is an aqueous solution containing deacylated gellan gum,

the linking substance of (ii) is one or both of calcium ion andmagnesium ion, and the second liquid is a liquid medium containing oneor both of calcium ion and magnesium ion.

[7] A production device for carrying out the production method of theabove-mentioned [1], comprising

a supply device for feeding out the first liquid,

a container for containing the second liquid and receiving the firstliquid fed out from the supply device, and

a nozzle part having a through-hole through which the first liquidpasses when fed out from the supply device into the container,

-   -   wherein        -   the through-hole has a cross-sectional area of 0.01 mm²-5.00            mm², and        -   the supply device is configured to feed out the first liquid            at the flow rate of not less than 1.7 mL/sec,            which is configured to be able to inject the first liquid            into the container at a flow rate of not less than 1.7            mL/sec by passing the first liquid through the through-hole            at the above-mentioned flow rate.            [8] The production device of the above-mentioned [7],            wherein

the nozzle part having the through-hole is provided as a part of thecontainer, and

the container has a body and a lid, and the nozzle part is provided inthe body or lid of the container such that the through-hole communicatesthe outside of the container and the inside of the container.

[9] The production device of the above-mentioned [8], wherein the supplydevice is a syringe,

the nozzle part is provided on the lid of the container, and a tubularcomponent for fitting a syringe tip protrudes from an outer surface ofthe lid at a container external side of the nozzle part.

[10] The production device of the above-mentioned [7], wherein thesupply device is a syringe, the nozzle part is an injection needlemounted on the syringe,

the lid of the container is provided with a penetratable part permittingpenetration of the injection needle from the outside of the container tothe inside of the container, and a tubular component for fitting aneedle base part of the injection needle further protrudes from an outersurface of the lid at a container external side of the penetratablepart. [11] A kit for carrying out the production method of theabove-mentioned [1], comprising at least a first container, a syringe,and a second container, wherein

the first container contains the first liquid of the above-mentioned[1],

the syringe is the syringe of the above-mentioned [3] functioning as asupply device for feeding out the first liquid,

the second container is the container (a) of the above-mentioned [2] forcontaining the second liquid of the above-mentioned [1] and, asdescribed in the above-mentioned [3], the nozzle part is provided on thelid of the container, and a tubular component for fitting a syringe tipprotrudes from an outer surface of the lid at a container external sideof the nozzle part.

[12] The kit of the above-mentioned [11], wherein the second containeris further equipped with a sealing lid free of a nozzle part andconfigured to be able to seal the inside of the body of the container,and the lid of the container (a) of the above-mentioned [2] and theabove-mentioned sealing lid can be compatibly mounted on an opening partof the body of the container.[13] The kit of the above-mentioned [11] or [12], wherein theabove-mentioned syringe is further equipped with a tubular component tobe inserted in the first container and used to suck the first liquid,said tubular component comprising

a thin-tube part having an outer diameter permitting insertion into thefirst container, and a length enabling suction of the first liquid fromthe first container, and

a connecting part at one end of the thin-tube part, mountable on the tipof the cylinder of the syringe.

Effect of the Invention

As described in the explanation of the above-mentioned Background Art,in a general stirring and mixing method including pouring a liquid(first liquid) containing a particular compound at a high concentrationinto a liquid medium (second liquid) containing a linking substancewhile stirring the medium, the structures are sometimes linked to form along string suspended in the mixture. The reason is that even if astirring bar is rotated at a high speed in the liquid medium, only thestirring bar moves certainly at a high speed, and the entire secondliquid (in particular, around liquid surface where the first liquid ispoured into) does not move as fast as the stirring bar.

To improve this, for example, a constitution in which the stirring baris moved closer to the junction where the first liquid is poured intoand stirs the mixture while cutting the junction is considered. Suchconstitution requires a special stirring apparatus which can be operatedin a sterile or sealed state to avoid biological contamination. Whenindividual production of a medium composition in a large number of smallcapacity containers is desired and the like, an open system is createdwhen a stirrer is inserted into the container, and therefore, a specialfacility to secure sterile condition such as clean room, clean booth andthe like needs to be installed. From such aspect, individual use of suchspecial stirring apparatus for each small capacity container isdifficult.

In contrast, the principle of the mixing method of the both liquids inthe production method and production device of the present invention iscompletely opposite to the principle of the above-mentioned conventionalmixing methods, and the second liquid is not stirred at a high speed butthe first liquid is injected at a flow rate Q (or flow velocity V) notless than a given value by passing the liquid through a through-holehaving a predetermined cross-sectional area S so that the first liquidis injected into the second liquid at the flow rate Q (or flow velocityV). In this way, the difference in the speed of the both liquids at thejunction where the both liquids meet can be certainly increased, wherebythe both liquids contact with impact and long string of the structurescan be sufficiently suppressed. Once fine structures are formed, evenwhen the fine structures gather in one part of the mixture, they can behomogeneously dispersed throughout the whole mixture in a container byfurther stirring.

In the present invention, the cross-sectional area S of the through-holein the nozzle part is limited to 0.01 mm²-5.00 mm² to set the firstliquid to a flow rate of not less than 1.7 mL/sec (L is liter). Thecross section of the through-hole is a section of the through-hole whenit is cut perpendicularly to the central axis from the inlet opening tothe outlet opening of the through-hole, and the cross-sectional area ofthe through-hole is an area of the cross-section of the through-hole.

The flow rate Q of the first liquid that passes through the outletopening of the through-hole, the cross-sectional area S of thethrough-hole, and the flow velocity V of the first liquid that passesthrough the outlet opening of the through-hole show a relationship ofQ=S×V.

First, when a through-hole having a large diameter exceeding theabove-mentioned cross-sectional area is used, the first liquid forming athick flow rushes into the second liquid. In such a thick flow, thefirst liquid in the center part thereof may not be able to contact thesecond liquid before stalling. In contrast, when the cross-sectionalarea of the through-hole is within the above-mentioned range, theinjection flow maintains a thin flow, and the first liquid in the centerpart thereof highly probably contacts the second liquid before stalling.Therefore, in the present invention, the cross-sectional area of thethrough-hole is limited to the above-mentioned range.

In the cultivation site of cell and the like, a supply device preferablefor practical use for feeding out the first liquid is a manual syringeas mentioned below. Therefore, feeding out at a flow rate capable ofachieving the preferable flow velocity cannot be certainly performedwith ease with a through-hole having a large diameter beyond theabove-mentioned cross-sectional area.

In the present invention, therefore, a through-hole having across-sectional area in the above-mentioned range is always used evenwhen a large amount of medium composition is produced at once in acontainer with large capacity. Where necessary, a plurality ofthrough-hole may be used to inject parallelly the first liquid in thesecond liquid, or a single through-hole may be used to inject the firstliquid in the second liquid plural times. The supply device in this casemay be one according to the discharge capability thereof, or supplydevices may be provided in parallel in the number of the through-holes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sectional drawing explaining the outline of the productionmethod and the constitution of the production device of the presentinvention. To divide and stress the regions, the section of the nozzlepart is hatched. The constituent elements shown by the symbols in FIG. 1are: A; the first liquid, B; the second liquid, A1; flow of injectedfirst liquid, 1; supply device, 2; nozzle part, 21; through-hole, 3;container.

FIG. 2 is a sectional drawing showing a specific embodiment of theproduction method and the production device of the present invention. Inthe embodiment of this drawing, the nozzle part belongs to the containerside, and the first liquid is configured to be injected in the center ofthe container. In this Figure, the sections of the body and the lid areshown as end surface drawings of the container, and the appearance ofthe syringe 1 as a supply device, rather than a section thereof, isshown (same in FIG. 3, FIG. 6). Hatching of the section is omitted (samein FIG. 3-FIG. 7).

FIG. 3 is a drawing showing other specific embodiment of the productionmethod and the production device of the present invention. In theembodiment of this drawing, the nozzle part belongs to the containerside, and the first liquid is configured to be injected in a positioneccentric from the center of the container.

FIG. 4 shows a partially enlarged view of the nozzle part in FIG. 2,FIG. 3. FIG. 4(a) is a top view of the nozzle part, FIG. 4(b) is apartially-enlarged sectional view of the nozzle part in FIG. 2, FIG. 3,and FIG. 4(c) is a side view of the upper part alone of FIG. 4(b), whichis seen from the X direction of the Figure.

FIG. 5 shows one embodiment of the lid of the container in theproduction device of the present invention. FIG. 5(a) is a top view ofthe lid, and FIG. 5(b) is a side view of the lid and is a sectional viewwhen the lid is cut along line Y-Y in FIG. 5(a).

FIG. 6 is a drawing showing other specific embodiment of the productionmethod and the production device of the present invention. In theembodiment of this drawing, the nozzle part is a component (needle)belonging to the syringe as a supply device, and the first liquid isconfigured to be injected in a position eccentric from the center of thecontainer.

FIG. 7 is a schematic showing one embodiment of the constitution of thekit of the present invention. In this Figure, the container and the lidare shown in sectional drawings. An appearance of the syringe 1 as asupply device rather than a section thereof is shown.

DESCRIPTION OF EMBODIMENTS

In the following, the production method of the present invention isexplained while explaining the constitution of the production device ofthe present invention. The explanation of the specific constitution ofthe production device of the present invention is also an explanation ofhow to practice the production method of the present inventionconcretely. The description of the production method of the presentinvention is also an explanation of the method of using the productiondevice of the present invention.

The production method of the present invention is preferably a method ofpreferably producing a liquid medium composition as described in theabove-mentioned patent document 1, and includes, as shown in FIG. 1,steps of passing a first liquid A comprising a particular compound ofthe following (i) through a through-hole 21 having a cross-sectionalarea S of 0.01 mm²-5.00 mm² formed in a nozzle part 2 at a flow rate Qof not less than 1.7 mL/sec, and injecting the first liquid with theabove-mentioned flow rate Q into a second liquid B comprising a linkingsubstance of the following (ii) to mix the both liquids.

Here, the particular compound of the above-mentioned (i) is a polymercompound having an anionic functional group, and capable of forming astructure by linking via a divalent metal cation, the structure beingcapable of suspending a cell or a tissue.

The linking substance of the above-mentioned (ii) is a divalent metalcation.

The particular compound, linking substance, and the first and secondliquids containing each are described in detail below.

By injecting at the above-mentioned flow rate Q, a liquid mediumcomposition containing a structure having a particular compound bondedvia a linking substance formed in a mixture of the above-mentioned bothliquids A, B but finely dispersed in the mixture can be obtained.

The flow rate Q of the first liquid A only needs to be not less than 1.7mL/sec. When the flow rate Q is larger relative to the through-holehaving the cross-sectional area S in the above-mentioned range, thefirst liquid violently rushes into the second liquid and suppresses along string of the structure.

While the upper limit of the flow rate is not particularly limited, fromthe aspect of the feed-out capability of the first liquid through-holein consideration of the cross-sectional area S, it is about 10 mL/sec,more suitably about 5 mL/sec for the actual operation.

The cross-sectional area S of the through-hole 21 in the nozzle part maybe 0.01 mm²-5.00 mm², more preferably 0.05 mm²-2.00 mm², particularlypreferably 0.10 mm²-0.70 mm². By limiting the cross-sectional area ofthe through-hole to fall within the above-mentioned range, preferablestirring results (namely, preferable dispersion results of thestructures) are obtained even when the amount of the first liquid to befed out from the supply device may vary somewhat.

The flow direction of the first liquid when it is injected into thesecond liquid is not particularly limited. As shown in FIG. 1, it may beinjected downwardly from the above, or may be lateral injection from theside, or upward injection from the below.

The production device of the present invention is a device configured tocarry out the production method of the present invention. As shown inFIG. 1, it is configured to have at least a supply device 1 and acontainer 3 in addition to the nozzle part 2 having the above-mentionedthrough-hole 21.

The above-mentioned supply device 1 is a device for feeding out thefirst liquid A, and configured to feed out the first liquid at theabove-mentioned flow rate Q (mm³/sec). The container 3 is a containerfor containing the second liquid B and receiving the first liquid fedout from the supply device 1, and forms a mixture of the both liquids(i.e., liquid medium composition to be produced).

The nozzle part 2 has a through-hole 21 having the above-mentionedcross-sectional area (mm²) through which the first liquid passes when itis fed out from the supply device into the container. The nozzle part 2may be a part belonging to the supply device 1, or a part belonging tothe body or lid of the container, or an independent coupling memberinterlaying between the supply device and the container and notbelonging to any member.

When the production device is used, the first liquid A is fed out at theabove-mentioned flow rate Q (mm³/sec) from the supply device 1, passesthrough a through-hole 21 having the above-mentioned cross-sectionalarea S (mm²) and formed in the nozzle part 2, and rushes into the secondliquid contained in the container 3. This series of operations carry outthe production method of the present invention, whereby a preferableliquid medium composition is obtained as mentioned above.

The volume ratio of the first liquid and the second liquid to be mixedis not particularly limited, and generally, (first liquid:secondliquid)=about (1:1)-(1:1000), preferably about (1:5)-(1:500), morepreferably about (1:10)-(1:100).

In an actual cultivation operation of cells and the like, to prepare afresh medium composition when in use, it is sometimes more preferable toindividually produce a medium composition in each container having asmall capacity of about 1-1000 mL, preferably about 10-200 mL, ratherthan producing a large amount of the medium composition at once in asingle large capacity container and dividing same in each small capacitycontainer for stock.

When a medium composition is individually produced in each containerhaving a small capacity mentioned above, specific capacity of the bothliquids to be mixed is preferably about 1 mL-1000 mL, more preferablyabout 10 mL-200 mL, of the second liquid to be placed in a container,into which about 0.01 mL-100 mL, more preferably about 0.1 mL-20 mL, ofthe first liquid is injected.

The supply device 1 and the capacity of the container 3 can beappropriately selected according to such combination.

The mixture of the first liquid and the second liquid may be a mediumcomposition as the production object, or may be further added with anadditive to produce a medium composition as the production object.

Conversely to the present invention, a medium composition as theproduction object can also be obtained by placing a small amount of thefirst liquid in a container and injecting a large amount of the secondliquid thereinto. However, since the first liquid is not easily admixeddue to high viscosity thereof, the amount of the liquid splashed andscattered on the wall surface and the like relative to the total amountof the liquid is non-negligible and the like, the contact state anddilution conditions are vastly different from those when a small amountof a liquid is injected at a high flow velocity into a large amount of aliquid. In addition, problems occur such as the time required forinjection, increase of pressure in the container, and the like.Therefore, in the present invention, injection of the first liquid intothe second liquid at the above-mentioned ratio is recommended.

The supply device may be any as long as it has discharge capabilitypermitting feeding out of the first liquid at the above-mentioned flowrate Q (not less than 1.7 mL/sec) and, for example, peristaltic pump,diaphragm pump, syringe and the like can be mentioned. A driving sourceof the supply device may be manual or may use a drive apparatus such asmotor and the like. Among others, a syringe shown in FIG. 2, FIG. 3,FIG. 6 (injection syringe) is a preferable supply device, since it has asimple constitution, is easily handled, is low costly (and therefore,may be disposable), and can achieve the flow rate even by manualpushing. For example, plastic disposable syringe such as Terumo syringefor prophylactic inoculation manufactured by Terumo (1 mL, model numberSS-01P)—Terumo syringe manufactured by Terumo (50 mL, model numberSS-50ESZ) and the like is suitable for producing a medium compositionfor each container mentioned above having a small capacity.

The “syringe” in the present invention is a device configured to have acylinder and a plunger (pusher). The present invention includes anembodiment using a syringe as it is and an embodiment further having aninjection needle mounted on a syringe tip.

The upper limit of the flow rate Q when the first liquid is fed out(volume that passes through a certain cross section in the flow per unittime: mm³/sec) is not particularly limited. Since a higher flow ratecauses higher flow velocity on passage through a through-hole in theabove-mentioned nozzle part, and the first liquid rushes into the secondliquid with impact, the structure can be more preferably subdivided anddispersed.

When a syringe is used as a supply device, as shown in FIG. 2, a plunger(piston part) 12 is pushed down with a pressing force (load) F capableof achieving the above-mentioned flow rate or flow velocity by passingthe first liquid A through a through-hole 21, and the first liquid Acontained in a cylinder 11 is fed out.

That is, “passing a first liquid through a through-hole having across-sectional area of 0.01 mm²-5.00 mm² formed in a nozzle part toachieve a flow rate of not less than 1.7 mL/sec” in the presentinvention means that pressing force F is applied on the first liquidthrough a supply device 1 so that the first liquid can pass through athrough-hole with the cross-sectional area at a flow rate of not lessthan 1.7 mL/sec, and the first liquid is extruded by the pressing forcethrough the through-hole at the discharge flow rate.

When a syringe is used as a supply device, the flow rate of the firstliquid fed out from the syringe can be adjusting the time required forthe whole stroke of the plunger. That is, a syringe is preferable sincethe flow rate (mm³/sec) of the object can be adjusted by controlling thetime necessary for the stroke of the plunger even when it is pushed byhuman power.

While the upper limit of the flow rate obtained by pushing the syringeby human power varies depending on the power of individual, it isgenerally about 5 mL/sec.

In an embodiment where a syringe with a small capacity is used, feedingout at a flow rate of not less than 1.7 mL/sec can be achieved when thecross-sectional area of the through-hole is 0.2 mm², syringe is Terumosyringe for prophylactic inoculation manufactured by Terumo (1 mL, modelnumber SS-01P), 1.7 mL of the first liquid is contained in the syringe,and whole moving (travelling) time T1 of plunger, which is required toextrude the whole first liquid, is not more than 1 second. When thesyringe is manually operated and in consideration of the force forpushing the plunger, the whole moving time T1 of the plunger is suitablyabout 0.2 second-1 second, and the flow rate obtained in this case is1.7 mL/sec-8.5 mL/sec.

In an embodiment where a syringe with a large capacity is used, feedingout at a flow rate of not less than 1.7 mL/sec can be achieved when thecross-sectional area of the through-hole is 0.2 mm² which is same asabove, syringe is Terumo syringe manufactured by Terumo (50 mL, modelnumber SS-50ESZ), 20 mL of the first liquid is contained in the syringe,the through-hole has the same bore diameter, and whole moving(travelling) time T2 of plunger, which is required to extrude the wholefirst liquid, is not more than 12 seconds. When the syringe is manuallyoperated and in consideration of the force for pushing the plunger, thewhole moving time T2 of the plunger is suitably about 2.5 seconds-12seconds, and the flow rate obtained in this case is 1.7 mL/sec-8.0mL/sec. The pressing force (load) necessary in this case is the same aswhen the above-mentioned small capacity syringe is used.

When the plunger is pushed by human power, it is difficult to maintainthe moving speed of the plunger strictly constant. However, when themoving time is within the above-mentioned range, the plunger can bemoved without deviating from the range even by human power, whereby theobject flow rate and the flow velocity at the outlet opening of thethrough-hole can be achieved without a large error.

The through-hole in the nozzle part may be any as long as it has across-sectional area S in the above-mentioned range and affords a flowrate in the above-mentioned range. While the through-hole preferably hasa straight pipe shape, it may have a draft angle for resin molding. Theflow A1 of the first liquid when fed out from the through-hole ispreferably a linear flow maintaining the cross-section of thethrough-hole to the possible extent rather than a spreading spray, whichin turn enables strong and deep injection into the second liquid. Theshape of the through-hole can be appropriately designed to achieve suchflow.

The shape of the cross section of the through-hole is not particularlylimited, and may be a circular shape, an ellipse shape, an oval shape, arectangle shape, an oblong shape, an irregular shape or the like. Sinceformation of the through-hole is easy, the shape of the cross section ofthe through-hole is preferably a circular shape. To intentionallydisturb and diffuse the flow of the injected first liquid, moreover, theshape of the cross section of the through-hole may be appropriatelydeformed.

The length of the through-hole is not particularly limited. When anozzle part is set on a container (body or lid), the length of thethrough-hole is preferably about 0.01 mm-10 mm. When the nozzle part isan injection needle of a syringe, the length of the through-hole ispreferably about 1.0 mm-100 mm.

The nozzle part 2 may be positioned at the center of the opening of thecontainer and the like, so that the first liquid A can be injected intothe center of the container 3 as shown in FIG. 2. When the first liquidis injected from the nozzle part into the center of the container, aneffect of moving around at the bottom of the container as diverged flowscan be obtained after the first liquid is deeply injected into thesecond liquid, as shown with arrows in the container in FIG. 2.

Alternatively, the nozzle part 2 may be positioned at a position offset(decentered) from the center of the opening of the container, so thatthe first liquid can be injected into a position offset from the centertoward the periphery of the container, as shown in FIG. 3. When thefirst liquid is injected into a position offset from the center towardthe periphery of the container, the first liquid enters deeply into thesecond liquid, makes a large turn at the bottom of the container, andaffords an effect of moving around as one flow in the container, asshown with arrows in the container in FIG. 3.

The position of the nozzle part can be appropriately determinedaccording to, for example, the viscosity of the first and secondliquids, the amount of liquid, the shape of the container and the like.

The container only needs to be able to contain the above-mentionedsecond liquid, receive injection of the first liquid and can contain amixture of these. When the first liquid is injected in an airtight stateinto a container containing the second liquid, the pressure in thecontainer increases. However, when the mixing ratio is about theabove-mentioned specific mixing ratio, an increase in the pressure doesnot pose a particular problem.

The capacity of the container is not particularly limited. A preferablecapacity is, for example, about 5 mL-1000 mL from the aspect of theabove-mentioned specific mixing ratio. While the cross sectional shapeof the container is not particularly limited, a circular shape ispreferable for achieving a smooth circulation due to convection duringmixing.

The ratio (L1/D1) of depth L1 of the inside of the container and borediameter D1 is not particularly limited. Even when containers have thesame capacity, when the ratio (L1/D1) is excessively high, the containerbecomes excessive long as a whole and is not preferable since convectionduring mixing is prevented. When the ratio (L1/D1) is excessively small,the container becomes a thin plate as a whole and is not preferablesince convection during mixing is prevented. A preferable ratio (L1/D1)is appropriately determined from about 0.5-30, preferably about 1.0-15,further preferably 2.0-8.0.

When the second liquid is contained in a container, a ratio of depth Zof the part occupied by the liquid (immersion part) and the borediameter d (Z/d) is not particularly limited, either, and isappropriately determined from about 0.5-20, preferably about 1.0-10,further preferably about 1.3-4.0.

In the embodiments of FIG. 2, FIG. 3, FIG. 6, the shape of the bottom ofthe container is depicted as a cone shape that becomes narrower towardthe deepest part. However, it may be a semi-spherical shape, a flatshape or the like, and is not particularly limited. From the aspects ofchanges in the convection state which are assumed due to the amount ofliquid and viscosity, and practical handling such as improvement ofrecovery rate by centrifugation of cells and the like to achieveefficient sedimentation and the like, a container having a cone-shapedbottom part that becomes narrower toward the deepest part (calledconical tube) of FIG. 2, FIG. 3, FIG. 6 is sometimes preferable.Examples of preferable container product include round tube 5 mL(352003), conical tube 15 mL (352095), conical tube 50 mL (352070),conical tube 175 mL (352076) and conical tube 225 mL (352075)manufactured by Japan Becton Dickinson, 15 mL centrifuge tube (MS-56150)and 50 mL centrifuge tube (MS-56500) manufactured by Sumitomo Bakelite,centrifuge tube 15 mL (2322-015), centrifuge tube 50 mL (2342-050) andcentrifuge tube 230 mL (2386-230) manufactured by Asahi Glass, and thelike.

The container 3 preferably has, as shown in FIG. 2, FIG. 3, FIG. 6, abody 31 having an opening, and a lid 32 to be mounted on the openingpart (the opening and wall part surrounding same), since the body of ageneral container as a physicochemical instrument can be utilized, andfrom the aspect of the liquid putting in and out operation.

The material of the container is not particularly limited. Examplesthereof include plastic such as polyethylene, polypropylene,polystyrene, polyethylene terephthalate, polycarbonate and the like,glass, metal and the like. The materials of the body and the lid of acontainer may be different from each other.

To facilitate the operation of the production device for the productionmethod, the nozzle part is preferably provided as a part (inflow part)of the container or a part (discharge part) of the supply device.

When the nozzle part is provided as a part of the container, the nozzlepart may be provided in the body of the container (not shown) or in thelid (FIG. 2, FIG. 3). The through-hole in these cases is a flow pathcommunicating the outside of the container and the inside of thecontainer by penetrating the nozzle part. The first liquid passesthrough the through-hole and injected into the second liquid in thecontainer.

Since the body of a general container can be utilized as aphysicochemical instrument, an embodiment having a nozzle part 2provided in a lid 32 as shown in FIG. 2, FIG. 3 is preferable. Anembodiment having a nozzle part 2 integrally formed in a lid 32 ispreferable, and an embodiment having separately-formed partsincorporated in the lid is also preferable.

In the embodiments shown in FIG. 2, FIG. 3, the supply device 1 is asyringe, and the nozzle part 2 is provided in the lid 32 of thecontainer 3. In both embodiments, a tubular component 33 for fitting atip (cylindrical tip) 11 a of the cylinder 11 of the syringe protrudesfrom the outer surface of the lid 32 of the nozzle part 2 toward theoutside of the container.

As mentioned above, nozzle part 2 may be located in the center of thelid (FIG. 2), or located offset from the center to the periphery of thelid (FIG. 3).

When the nozzle part is offset, the opening shape of the container has acircular shape, the radius of the opening is R (mm), the distance ofoffset from the center to the periphery of the opening is r (mm), and aratio of r relative to R [(r/R)×100] is about 40%-80%, the effect of theoffset becomes remarkable. When the offset of the nozzle part isexcessively large, the flow of the first liquid is too close to thecontainer wall to possibly inhibit suitable stirring.

The distance from the nozzle tip to the liquid surface is preferablyfrom 0 mm to not more than 200 mm, more preferably not more than 150 mm,further preferably not more than 85 mm.

FIG. 4 is a partially-enlarged view showing in detail the nozzle partprovided in the lid and the structure outside thereof in the embodimentsof FIG. 2, FIG. 3.

As shown in FIG. 4(b), the tip (inside the container) of the nozzle part2 ensures the length of the through-hole 21 and protrudes into theinside of the container so that the first liquid can leave thethrough-hole as a linear flow maintaining the cross section of thethrough-hole to the possible extent. A space may be formed between thetip of the nozzle part and the liquid surface of the second liquidcontained in the container, and the tip of the nozzle part may extendlong to contact the second liquid.

As a preferable option, in the embodiment of FIG. 4(a)-(c), the tip ofthe tubular component 33 protruding from the outer surface of the lidhas protrusion parts 34, 35 protruding on the side, which are to beengaged with female threads (internal screw) on the tip of a luer-locktype syringe to fix the syringe by screwing therein. On the bottom partsof the protrusion parts 34, 35 are provided gradient 34 a, 35 a capableof fitting the female threads on the tip of the syringe, as shown inFIG. 4(c).

The coupling structure and sealability between the body 31 and the lid32 of the container, the coupling structure between the tip 11 a of thesyringe and the tubular component 33 of the lid, and fitting andsealability of the tip 11 a and the inner surface 33 a of the tubularcomponent can be set as appropriate.

FIG. 5 shows an embodiment in which a practical structure is added tothe detail of the lid shown in FIG. 3. The embodiment of this Figure hascompatibility with the lid originally attached to the container so thata commercially available container for physicochemical experiments canbe utilized as the body.

As shown in FIG. 5(b), a screw ridge 36 is formed in the inside of thelid 32 so that threaded engagement (screw together) with an opening partof the body of a commercially available container for physicochemicalexperiments can be achieved. In addition, an annular sealing part 37 forsealing the opening when the lid is threadedly engaged with the openingpart of the container protrudes from the innermost surface of the insideof the lid 32. As shown in FIG. 5(a), a knurling 38 similar to theoriginal lid is formed on the side of the outer circumference of thelid, which prevents slip in turning the lid with a hand.

In the embodiment shown in FIG. 6, a nozzle part is formed as a part ofa supply device. In the embodiment of this Figure, a supply device 1 isa syringe, a nozzle part is an injection needle 13 mounted on the tip ofthe syringe, and an injection needle 13 has a needle base part 13 a anda needle tube part 13 b. The needle tube part 13 a does not always needto be sharp.

In the embodiment of this Figure, the conduit in the inside of theneedle tube part is the through-hole in the present invention, and thecross-sectional area thereof is 0.01 mm²-5.00 mm². In the embodiment ofthis Figure, the tip of the needle tube part enters into the inside ofthe second liquid contained in the container.

The lid 32 of the container is provided with a penetratable part 39permitting penetration of a needle tube part 13 b of the injectionneedle 13 from the outside of the container into the container. Atubular component 33 for fitting a needle base part 13 a of theinjection needle protrudes from the outer surface of the lid 32 towardthe outside of the container of the penetratable part 39. While thetubular component 33 is not essential, it is a preferable structure forretaining the syringe when in use.

The penetratable part 39 only needs to be formed to permit passage ofthe needle tube part when in use, and may be a through-hole matchingwith the outer diameter of the needle tube part or a weakened part orthin film part through which a needle tube part can penetrate.

When the first liquid is injected into the second liquid by theproduction method of the present invention, the second liquid may bestirred and the first liquid may be injected into the stirring secondliquid.

Examples of the stirring method include a method of applying manualshaking, mechanical shaking (linear reciprocating motion, eccentricrotating motion and 8-shaped rotating motion), ultrasonicationtrembling, vortex stirring and the like to the container to stir thesecond liquid in the container.

The first liquid contains, as a particular compound, a polymer compoundhaving an anionic functional group and capable of forming a structurethat can suspend cells or tissues by binding via a divalent metalcation.

As the anionic functional group, carboxy group, sulfo group, phosphategroup and a salt thereof can be mentioned, with preference given tocarboxy group or a salt thereof. A polymer compound to be used in thepresent invention may contain one or more kinds selected from the groupof the aforementioned anionic functional groups.

Specific preferable examples of the polymer compound to be used in thepresent invention include, but are not limited to, polysaccharideswherein not less than 10 monosaccharides (e.g., triose, tetrose,pentose, hexsauce, heptose etc.) are polymerized, more preferably,acidic polysaccharides having an anionic functional group. The acidicpolysaccharides here is not particularly limited as long as it has ananionic functional group in the structure thereof, and includes, forexample, polysaccharides having a uronic acid (e.g., glucuronic acid,iduronic acid, galacturonic acid, mannuronic acid), polysaccharideshaving a sulfate group or phosphate group in a part of the structurethereof, and polysaccharides having the both structures, and includesnot only naturally-obtained polysaccharides but also polysaccharidesproduced by microorganisms, polysaccharides produced by geneticengineering, and polysaccharides artificially synthesized using anenzyme. More specifically, examples thereof include polymer compoundscomposed of one or two or more kinds from the group consisting ofhyaluronic acid, gellan gum, deacylated gellan gum (hereinaftersometimes to be referred to as DAG), rhamsan gum, diutan gum, xanthangum, carageenan, xanthan gum, hexuronic acid, fucoidan, pectin, pecticacid, pectinic acid, heparan sulfate, heparin, heparitin sulfate,keratosulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfateand a salt thereof. Polysaccharides are preferably hyaluronic acid, DAG,diutan gum, xanthan gum, carageenan or a salt thereof, more preferablyDAG or a salt thereof. Phosphorylated DAG can also be used. Thephosphorylation can be performed by a known method.

The salt here includes, for example, salts with alkali metal such aslithium, sodium, potassium; salts with alkaline earth metals such ascalcium, barium, magnesium; and salts with aluminum, zinc, copper, iron,ammonium, organic base and amino acid and the like.

The weight average molecular weight of these polymer compounds(polysaccharides etc.) is preferably 10,000 to 50,000,000, morepreferably 100,000 to 20,000,000, still more preferably 1,000,000 to10,000,000. For example, the molecular weight can be measured based onpullulan by gel penetration chromatography (GPC).

In the present invention, plural kinds (preferably two kinds) of theabove-mentioned polysaccharides having an anionic functional group canbe used in combination. The kind of the combination of thepolysaccharides is not particularly limited as long as theaforementioned structure is formed in a liquid medium by linking via adivalent metal cation. Preferably, the combination includes at least DAGor a salt thereof. That is, a preferable combination of polysaccharidescontains DAG or a salt thereof, and polysaccharides other than DAG and asalt thereof (e.g., xanthan gum, alginic acid, carageenan, diutan gum,methylcellulose, locust bean gum or a salt thereof). Examples ofspecific combination of polysaccharides include, but are not limited to,DAG and rhamsan gum, DAG and diutan gum, DAG and xanthan gum, DAG andcarageenan, DAG and xanthan gum, DAG and locust bean gum, DAG andκ-carageenan, DAG and sodium alginate, DAG and methylcellulose and thelike.

The deacylated gellan gum is a linear high molecular weightpolysaccharide containing 4 molecules of sugars of 1-3 bonded glucose,1-4 bonded glucuronic acid, 1-4 bonded glucose and 1-4 bonded rhamnoseas the constituent unit, which is a polysaccharide of the followingformula (I) wherein R₁, R₂ are each a hydrogen atom, and n is an integerof two or more. R₁ may contain a glyceryl group, R₂ may contain anacetyl group, and the content of the acetyl group and glyceryl group ispreferably not more than 10%, more preferably not more than 1%.

The particular compound may be obtained by a chemical synthesis method.When the particular compound is a naturally-occurring substance, it maybe obtained from various plants, various animals, various microorganismscontaining the compound by extraction, separation and purification byconventional techniques. For example, gellan gum can be produced byculturing producing microorganisms in a fermentation medium, recoveringmucous products produced outside the bacterial cells by a generalpurification method, and, after the processes of drying, pulverizing andthe like, powderizing the products. In the case of deacylated gellangum, an alkali treatment should be applied when the mucous products arerecovered, to deacylate the glyceryl group and the acetyl group bondedto 1-3 bonded glucose residue, and then the given products arerecovered. Examples of the gellan gum-producing microorganism include,but are not limited to, Sphingomonas elodea and microorganism obtainedby altering the gene of Sphingomonas elodea.

In the case of deacylated gellan gum, commercially available products,for example, “KELCAOGEL (registered trade mark of CP Kelco) CG-LA”manufactured by SANSHO Co., Ltd., “KELCOGEL (registered trade mark of CPKelco)” manufactured by San-Ei Gen F.F.I., Inc. and the like can beused. As a native type gellan gum, “KELCOGEL (registered trade mark ofCP Kelco) HT” manufactured by San-Ei Gen F.F.I., Inc. and the like canbe used.

The first liquid is generally a solution of the particular compound.While the solvent for the solution is not particularly limited as longas it can dissolve the particular compound, it is generally water orhydrophilic solvent, preferably water. In a preferable embodiment,therefore, the first liquid is an aqueous solution of the particularcompound.

The concentration of the particular compound contained in the firstliquid is not particularly limited as long as, upon mixing with thesecond liquid, the particular compounds are linked via a divalent metalcation to form a structure capable of suspending cells or tissues in themixture, the structure is uniformly dispersed in the mixture, andfurther, the finally-obtained liquid medium composition containing thestructure can cultivate the cells or tissue in suspension. Theconcentration of the particular compound in the first liquid can becalculated from the concentration of the particular compound in themedium composition capable of culturing cells or tissues in suspensionand the ratio of the volume of the first liquid to the volume of themedium composition obtained as the final product, as described in detailbelow. For example, when the first liquid with volume V₁ and the secondliquid with volume V₂ are mixed to finally obtain a liquid mediumcomposition with volume V₁+V₂, the concentration C % (w/v) of theparticular compound in the liquid medium composition can be achieved bysetting the concentration of the particular compound in the first liquidto C×(V₁+V₂)/V₁% (w/v). In a specific embodiment, when DAG or a saltthereof is used as a particular compound, the concentration of DAG inthe first liquid is, for example, 0.02-2.5% (w/v), preferably 0.1-2.0%(w/v), more preferably 0.5-1.5% (w/v). When the concentration of DAGexceeds 2.5% (w/v), DAG is not easily dissolved in the solvent from theviewpoint of solubility, the structures are topically formed upon mixingof the first liquid and the second liquid and entangled in a mass,thereby increasing the risk of difficult dispersing in the mediumcomposition. On the other hand, when the concentration of DAG is lowerthan 0.02% (w/v), the volume of the first liquid necessary for producingthe object medium composition becomes large. As a result, when a generalliquid medium is adopted as the second liquid, the components derivedfrom the liquid medium are drastically diluted when mixed with the firstliquid.

The concentration of the divalent metal cation in the first liquid needsto be lower than the concentration at which the particular compoundforms the structure in the first liquid. Examples of the divalent metalcation include calcium ion, magnesium ion, zinc ion, manganese ion,ferrous ion, copper ion and the like. Particularly, one or both ofcalcium ion and magnesium ion (hereinafter to be also referred to as“calcium ion and/or magnesium ion”) contributes to the structureformation of a particular compound such as DAG and the like.

The first liquid may contain factors other than a particular compoundand a solvent. Examples of the factor include, but are not limited to,physiologically acceptable buffering agent, salt, and isotonic agent.

The first liquid can be prepared by adding a particular compound to theabove-mentioned solvent (e.g., water), stirring the mixture at atemperature capable of dissolving the particular compound (e.g., notless than 60° C., not less than 80° C., not less than 90° C.), anddissolving until a transparent state is formed. Using DAG subjected todivalent metal cation-exclusion treatment as mentioned above, adissolution operation is easy since it is dissolved in water withoutrequiring heating. Where necessary, the obtained solution of theparticular compound is subjected to a divalent metal cation-exclusiontreatment to render the concentration of the divalent metal cation inthe solution lower than the structure-forming concentration. Wherenecessary, a factor other than the particular compound may be added tothe solvent in advance, or a factor other than the particular compoundmay be added to the obtained solution of the particular compound. Thefirst liquid is preferably sterilized. Examples of the method of thesterilization include, but are not limited to, autoclave, filtrationsterilization and the like.

The second liquid contains a divalent metal cation as a linkingsubstance. Examples of the divalent metal cation include calcium ion,magnesium ion, zinc ion, manganese ion, ferrous ion, copper ion and thelike. While the kind of the divalent metal cation is not particularlylimited as long as the particular compound in the first liquid is bondedvia a divalent metal cation to form a structure capable of suspendingcells or tissues, with preference given to calcium ion.

The second liquid is generally a solution of a linking substance (i.e.,divalent metal cation). While the solvent for the solution is notparticularly limited as long as it can dissolve the particular compound,it is generally water or hydrophilic solvent, preferably water. In apreferable embodiment, therefore, the second liquid is an aqueoussolution of a linking substance (i.e., divalent metal cation).

The second liquid contains a divalent metal cation at a concentrationsufficient for the particular compound in the first liquid to form thestructure when the first liquid and the second liquid are mixed. Forexample, when calcium ion or magnesium ion is used as the linkingsubstance, the concentration of the calcium ion or magnesium ion in thesecond liquid is generally not less than 0.001 mM, preferably not lessthan 0.01 mM. The upper limit of the concentration of the divalent metalcation in the second liquid is theoretically the concentration of asaturated solution (i.e., solubility). When the concentration of thedivalent metal cation is too high, the cells and the like to be culturedin the produced medium composition may be adversely influenced.Therefore, for example, when calcium ion or magnesium ion is used as thelinking substance, the upper limit of the concentration of the calciumion or magnesium ion in the second liquid is generally not more than 100mM, preferably not more than 10 mM. When the second liquid contains bothcalcium ion and magnesium ion, the total concentration of these ions isnot more than 100 mM, preferably not more than 10 mM.

The second liquid may contain factors other than a linking substance(i.e., divalent metal cation) and the solvent. Examples of the factorinclude medium constituent components suitable for culturing theintended cells. Examples of the medium constituent component include,but are not limited to, buffering agent (carbonate buffer, phosphatebuffer, HEPES etc.), inorganic salts (NaCl etc.), various amino acids,various vitamins (choline, folic acid etc.), saccharides (glucose etc.),antioxidants (monothioglycerol etc.), pyruvic acid, fatty acids, serum,antibiotics, insulin, transferrin, lactoferrin, cholesterol, variouscytokines, various hormones, various growth factors, variousextracellular matrices, and the like. The second liquid is preferablysterilized. Examples of the sterilization method include, but are notlimited to, autoclave, filtration sterilization and the like.

In a preferable embodiment, the second liquid is a liquid mediumcontaining a divalent metal cation (preferably, calcium ion and/ormagnesium ion) at the structure-forming concentration. That is, thesecond liquid contains, in addition to a divalent metal cation(preferably, calcium ion and/or magnesium ion) at the structure-formingconcentration and water, medium constituent components suitable forculturing the intended cells. The concentration of the calcium ion in agenerally-used liquid medium for cell culture is about 0.1-2.0 mM, andthe concentration of the magnesium ion is about 0.1-1.0 mM, which aresufficient for formation of the structure by a particular compound suchas DAG and the like.

In this embodiment, the intended liquid medium composition containing astructure in which particular compounds contained in the first liquidare linked via a linking substance contained in the second liquid can beobtained immediately by mixing the first liquid and the second liquidaccording to the production method of the present invention.

The second liquid may not contain a part or the whole of the mediumconstituent component for cell culture. In this case, the intendedliquid medium composition can be obtained by, in the production methodof the present invention, mixing the first liquid and the second liquidto give a mixture containing a structure in which particular compoundscontained in the first liquid are linked via a linking substancecontained in the second liquid and adding a part or the whole of theabove-mentioned liquid medium constituent component for cell culture tothe mixture.

The liquid medium composition that can be obtained by the productionmethod of the present invention contains a structure in which particularcompounds contained in the first liquid are linked via a linkingsubstance (i.e., divalent metal cation) contained in the second liquid,wherein the structures are uniformly dispersed in the mediumcomposition. Therefore, using the medium composition, cells and tissuescan be cultured while maintaining the suspended state.

The type of organism from which cells or tissues to be cultured arederived is not particularly limited, and may be not only animals(insect, fish, amphibian, reptiles, birds, pancrustacea, hexapoda,mammals and the like) but also plants.

In one embodiment, the cell to be cultured is an anchorage dependentcell. Using the liquid medium composition that can be obtained by theproduction method of the present invention, anchorage dependent cellscan be cultured while maintaining a suspended state, without using acarrier to be the anchorage.

Suspending of cells and/or tissues in the present invention refers to astate where cells and/or tissues may contact the bottom surface but donot adhere to a culture container (non-adhesive). Furthermore, in thepresent invention, when the cells and/or tissues are proliferated,differentiated or maintained, the state where the cells and/or tissuesare uniformly dispersed and suspended in the liquid medium compositionin the absence of a pressure on or vibration of the liquid mediumcomposition from the outside or shaking, rotating operation and the likeis referred to as “static suspension”, and culturing of the cells and/ortissues in such condition is referred to as “static suspension culture”.In the “static suspension”, the duration of suspending includes not lessthan 5 min, not less than 1 hr, not less than 24 hr, not less than 48hr, not less than 7 days and the like, though the duration is notlimited thereto as long as the suspended state is maintained.

The medium composition that can be obtained by the production method ofthe present invention permits static suspension of cells and/or tissuesat least on one point in the temperature range (e.g., 0-40° C.) capableof maintaining or culturing cells or tissues. The medium composition tobe used in the present invention permits static suspension of cellsand/or tissues at least at one point in the temperature range ofpreferably 25-37° C., most preferably 37° C.

Whether or not static suspension is possible can be evaluated by, forexample, uniformly dispersing the cells to be cultured in a mediumcomposition to be evaluated at a concentration of 2×10⁴ cells/ml,injecting 10 ml thereof in a 15 ml conical tube, standing the tube forat least not less than 5 min (e.g., not less than 1 hr, not less than 24hr, not less than 48 hr, not less than 7 days) at 37° C., and observingwhether the suspended state of the cells is maintained. When not lessthan 70% of the total cells are in a suspended state, it is concludedthat the suspended state was maintained. Polystyrene beads (Size 500-600μm, manufactured by Polysciences Inc.) may be used for evaluationinstead of the cells

In a preferable embodiment, in the liquid medium composition that can beobtained by the production method of the present invention, theviscosity thereof is not substantially increased by the containedabove-mentioned structure, since it contains the above-mentionedstructure. The terms “not substantially increasing the viscosity of theliquid” means that the viscosity of the liquid does not exceed 8 mPa·s.In this case, the viscosity of the liquid (that is, the viscosity of theliquid medium composition that can be obtained by the production methodof the present invention) is not more than 8 mPa·s, preferably not morethan 4 mPa·s, more preferably not more than 2 mPa·s at 37° C. Theviscosity of the liquid containing the structure can be measured under37° C. conditions and using an E-type viscosity meter (manufactured byToki Sangyo Co., Ltd., TV-22 type viscosity meter, model: TVE-22 L, cornroter: standard roter 1° 34′×R24, rotating speed 100 rpm).

The concentration of the particular compound in a liquid mediumcomposition that can be obtained by the production method of the presentinvention depends on the kind of the particular compound, and can beappropriately determined within the range where the particular compoundcan form the aforementioned structure in a liquid medium composition,and can uniformly suspend (preferably statically suspend) the cellsand/or tissues (preferably, without substantially increasing theviscosity of the liquid). In the case of DAG, it is 0.001% to 1.0%(w/v), preferably 0.003% to 0.5% (w/v), more preferably 0.005% to 0.3%(w/v), further preferably 0.01% to 0.05% (w/v), most preferably 0.01% to0.03% (w/v). In the case of xanthan gum, it is 0.001% to 5.0% (w/v),preferably 0.01% to 1.0% (w/v), more preferably 0.05% to 0.5% (w/v),most preferably 0.1% to 0.2% (w/v). In the case of κ-carrageenan andlocust bean gum mixture system, the total of the both compounds is0.001% to 5.0% (w/v), preferably 0.005% to 1.0% (w/v), more preferably0.01% to 0.1% (w/v), most preferably 0.03% to 0.05% (w/v). In the caseof a native type gellan gum, it is 0.05% to 1.0% (w/v), preferably 0.05%to 0.1% (w/v).

When plural kinds (preferably two kinds) of the above-mentionedpolysaccharides are used in combination as a particular compound, theconcentration of the polysaccharides can form the aforementionedstructure in a liquid medium composition, and can uniformly suspend(preferably statically suspend) the cells and/or tissues (preferablywithout substantially increasing the viscosity of the liquid). Forexample, when a combination of DAG or a salt thereof and polysaccharideother than DAG and a salt thereof is used, the concentration of DAG or asalt thereof is, for example, 0.005-0.02% (w/v), preferably 0.01-0.02%(w/v), and the concentration of polysaccharide other than DAG and a saltthereof is, for example, 0.0001-0.4% (w/v), preferably 0.005-0.4% (w/v),more preferably 0.1-0.4% (w/v). Specific examples of the combination ofthe concentration range include the following.

DAG or a salt thereof: 0.005-0.02% (preferably 0.01-0.02%) (w/v)polysaccharide other than DAGxanthan gum: 0.1-0.4% (w/v)sodium alginate: 0.0001-0.4% (w/v) (preferably 0.1-0.4% (w/v))native gellan gum: 0.0001-0.4% (w/v)locust bean gum: 0.1-0.4% (w/v)methylcellulose: 0.1-0.4% (w/v) (preferably 0.2-0.4% (w/v))carageenan: 0.05-0.1% (w/v)diutan gum: 0.05-0.1% (w/v)

The concentration can be calculated by the following formula.

Concentration [% (w/v)]=weight (g) of particular compound/volume (mL) ofmedium composition×100

In a preferable embodiment, DAG or a salt thereof is used as theparticular compound, and calcium ion is used as the linking substance.The first liquid is an aqueous solution of DAG or a salt thereof. Theconcentration of DAG in the aqueous solution is generally 0.05-1.5%(w/v), preferably 0.1-1.2% (w/v), more preferably 0.5-1.0% (w/v). Thesecond liquid is a liquid medium containing calcium ion. Theconcentration of calcium ion in the liquid medium is a concentration atwhich DAG forms a structure, and is generally about 0.1-2.0 mM. Thevolume ratio of the first liquid and the second liquid to be mixed is(first liquid:second liquid)=(0.5-10:100), preferably (1-5:100), morepreferably (1.5-3:100). The volume of the first liquid is, for example,0.1-20 mL, preferably 0.3-12 mL, more preferably 0.6-6 mL. The volume ofthe second liquid is, for example, 1-1000 mL, preferably 10-500 mL, morepreferably 40-200 mL. The concentration of DAG in the resulting liquidmedium composition is preferably 0.01%-0.05% (w/v), most preferably0.01%-0.03% (w/v).

Since the liquid medium composition contains a structure, in which DAGis linked via a calcium ion, uniformly dispersed therein, the cellsand/or tissues can be uniformly suspended (preferably staticallysuspended) without substantially increasing the viscosity.

Using the liquid medium composition obtained by the production method ofthe present invention, cells and/or tissues can be cultured in asuspended state without an operation such as shaking, rotation and thelike having a risk of causing damage and loss of functions of cells andtissues. Furthermore, using the medium composition, the medium can beexchanged easily during culture, and the cultured cells and/or tissuescan also be recovered easily. Using the medium composition, adhesivecells can be prepared efficiently in a large amount without impairingthe function thereof, since cells conventionally required to be culturedon a plate in a single layer in an adhered state to a cell container canbe cultured in a suspended state.

Now, the kit of the present invention is explained. The kit of thepresent invention is, as shown in FIG. 7, a set of assembly of theconstituent elements for practicing the above-mentioned productionmethod of the present invention.

As shown in FIG. 7, the kit of the present invention contains the firstcontainer K1 containing the above-mentioned first liquid, and isconfigured to at least comprise syringe K2 as a supply device 1 forfeeding out the first liquid explained above and the second container K3as a container 3 explained by reference to FIG. 2-FIG. 5. The secondcontainer K3 is, as explained above as regards container 3, a containerhaving lid K32 with a nozzle part and body K31, and further has, on thecontainer outside side of the nozzle part, a tubular component to fitthe tip of the syringe, protruding from the outer surface of the lid.

The first container K1 is not limited as to the material and size aslong as it can contain the above-mentioned first liquid only in anamount required by a user and can deliver same to the user. The firstcontainer K1 is preferably in an embodiment having a container body anda lid for closing the opening of the container body, and the lid ispreferably in an embodiment threadedly engaged with the container body.

The second liquid may be provided in a kit, or may be a liquid mediumgenerally used by a user.

In a preferable embodiment of the kit of the present invention, as shownin FIG. 7, the second container K3 is further equipped with a sealinglid K32 without a nozzle part, which is configured to be able to sealthe inside of the body K31 of the container. The sealing lid K32 iscompatible with the lid K32 of the container and can be mounted on theopening part of the body K31 of the container.

Provision of the sealing lid K32 enables production of a liquid mediumcomposition in the body K31 of the container by using lid K32 having anozzle part, and sealing the liquid medium composition in body K31 ofthe container.

When the body K31 of the container is a commercially available product,the sealing lid K32 may be a lid originally attached to the containerbody of the commercially available product.

In a preferable embodiment of the kit of the present invention, as shownin FIG. 7, a tubular component K21 is further equipped, which can bemounted on the tip of the above-mentioned syringe K2. The tubularcomponent is a suction tube used by inserting in the first container toaspirate the first liquid into the syringe. The tubular component has athin-tube part having an outer diameter permitting insertion into thefirst container K1 and a length permitting aspiration of the firstliquid from the inside of the first container, as well as a connectingpart mountable on the tip of the cylinder of the syringe, at one end ofthe thin-tube part. The tubular component may be an injection needle,but an economical resin molded part is preferable.

EXAMPLES

Various Experimental Examples evaluating the production device andproduction method of the present invention are shown below. The“Comparative Example” in each of the following Experimental Examples isan Experimental Example conducted according to the technical idea of thepresent invention to investigate preferable numerical values andconditions of the present invention, and does not refer to the PriorArt.

The concentration of divalent cation in the medium used in each test isas shown in the following Table 1.

TABLE 1 ion concentration calcium magnesium copper iron zinc medium [mM][mM] [nM] [μM] [μM] DMEM (high glucose) 1.8 0.81 — — — DMEM-F12 1.1 0.715.2 1.6 1.5 Ham's F12 0.3 0.6  10 3.0 3.0 EMEM 1.8 0.81 — — — RPMI 0.90.41 — — —

Experimental Example 1: Test for Evaluation of the Production Device andProduction Method of the Present Invention

One embodiment of the production device of the present invention wasproduced, the production method of the present invention was carried outusing same, and the results of the test observing the dispersion stateof the structure in the obtained medium composition are shown in thefollowing. As a Comparative Example, the test results when the flow rateof the injection of the first liquid was decreased are shown.

In Examples 1-9 and Comparative Examples 1-16, a particular compound wasdeacylated gellan gum, the first liquid was a deacylated gellan gumsolution, and a liquid medium containing calcium ion and magnesium ionas a linking substance to link the deacylated gellan gum to form thestructure was the second liquid.

[Specifications of Production Device]

In Examples 1-9 and Comparative Examples 1-16, a production device ofthe type shown in FIG. 3 was produced.

As a container, the body part of a commercially available conical tubewith capacity 225 mL (225000 mm³) or capacity 50 mL (50000 mm³)(manufactured by SUMITOMO BAKELITE) was used, and a lid matching same asshown in FIG. 5 was produced. The bore diameter of the through-hole was0.5 mm. Therefore, the cross-sectional area of the through-hole wasabout 0.2 mm².

As a supply device, a disposable syringe with capacity 5 mL (5000 mm³)or capacity 1 mL (1000 mm³) was used, and a tip of the cylinder of thesyringe could be coupled by being fitted into the cylindrical part 33protruding from the outside of the lid.

[Preparation of First Liquid]

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.)was suspended in ultra pure water (Milli-Q water) to 0.75% (w/v),dissolved by stirring with heating at 90° C., and this aqueous solutionwas autoclave sterilized at 121° C. for 20 min.

[Preparation of Liquid Medium Composition Product]

As the second liquid, Dulbecco's Modified Eagles's Medium (DMEM highglucose, manufactured by Wako Pure Chemical Industries, Ltd.) (200 mL)was contained in a 225 mL conical tube (manufactured by SUMITOMOBAKELITE) and cooled to 4° C. The concentration of calcium ion in DMEMwas 1.80 mM, and the magnesium ion concentration was 0.8 mM.

The cap of the conical tube filled with the second liquid cooled to 4°C. was removed, and a lid produced as the production device of thepresent invention was mounted.

The tip of the disposable syringe filled with the above-mentionedsterilized aqueous deacylated gellan gum solution (4 mL, 4000 mm³) wasfitted into the cylindrical part of the lid to form connection, wherebythe production device of the present invention, which is of the typeshown in FIG. 3, was constituted.

In this state, plunger of the syringe was pushed by human power, and thedeacylated aqueous gellan gum solution in the syringe was injected intothe container by moving same at the constant speed to give a mediumcomposition.

The conditions of concentration and volume of each liquid, flow rate ofthe first liquid injection and the like were changed to give Examples1-9 and Comparative Examples 1-16. The flow rate of the first liquid ineach Example was achieved by adjusting the moving time of the syringeplunger. In Examples 3, 5, 8, and Comparative Examples 3, 5, 10, 14, 16,the first liquid was injected into the second liquid in a stirring statein a whirlpool manner by using a vortex mixer (2500 rpm).

After injection, the adapter cap was changed to a cap of a conical tube,and the tube was tightly sealed.

[Evaluation]

Polystyrene beads (diameter 500-600 μm, manufactured by PolysciencesInc.) for simulating suspended cells were added to the produced mediumcomposition and stirred, and the dispersed state of the beads in theliquid was confirmed by visual observation 10 min after discontinuationof the stirring.

When the structure is appropriately finely dispersed in the liquid, thebeads are also dispersed and remain suspended in the liquid. On theother hand, when dispersion of the structure is not sufficient, thebeads also settle down accordingly.

When the structure is appropriately finely dispersed in the liquid, thestructure cannot be recognized by visual observation. On the other hand,when the structure forms a string or gathers, the structure can berecognized by visual observation as a material scattering the light, ora transparent amorphous string material visually observed due to therefraction of light.

The test conditions, dispersion state of beads in the resultingproducts, and the state of the structures in Examples 1-9 andComparative Examples 1-16 are shown in Table 2.

In Table 2, the dispersion state of the beads is shown with ◯ forpreferably dispersed and suspended state, Δ for dispersed but partlysedimented state, and x for the state of sedimentation of all beads.

Whether the structure cannot be visually recognized (considered to beappropriately finely dispersed), or is string or mass is shown with ◯,Δ, x as follows.

◯ shows that the structure cannot be visually recognized as a suspendedmaterial, as in commercially available liquid medium composition FCeM(registered trade mark).

Δ is a material that scatters light when held to light, or transparentamorphous string material permitting visual observation of suspendedstate due to the refraction of light.

x is a state permitting visual observation of suspended state orformation of sediment of the structure as a clear fibrous precipitate.

TABLE 2 second liquid B first liquid A evaluation results amount amountflow suspended state added stirring concentration added rate state of ofkind [mL] [rpm] [%] [mL] [mL/s] beads structure Ex. 1 DMEM 200 0 0.75 45.0 ∘ ∘ Ex. 2 DMEM 200 0 0.5 6 4.3 ∘ ∘ Ex. 3 DMEM 200 2500 1 3 4.3 ∘ ∘Ex. 4 DMEM 200 0 1 3 4.3 ∘ ∘ Ex. 5 DMEM 40 2500 1 0.6 3.0 ∘ ∘ Ex. 6 DMEM40 0 1 0.6 3.0 ∘ ∘ Ex. 7 DMEM 200 0 0.75 4 2.0 ∘ ∘ Ex. 8 DMEM 200 2500 13 2.0 ∘ ∘ Ex. 9 DMEM 200 0 1 3 1.7 ∘ ∘ Com. DMEM 40 0 1 0.6 1.5 Δ Δ Ex.1 Com. DMEM 200 0 0.5 6 1.3 ∘ Δ Ex. 2 Com. DMEM 40 2500 1 0.6 1.2 Δ ΔEx. 3 Com. DMEM 40 0 1 0.6 1.2 Δ Δ Ex. 4 Com. DMEM 200 2500 1 3 1.0 Δ xEx. 5 Com. DMEM 200 0 0.75 4 0.78 Δ Δ Ex. 6 Com. DMEM 200 0 0.5 6 0.76 ∘Δ Ex. 7 Com. DMEM 200 0 0.5 6 0.66 Δ x Ex. 8 Com. DMEM 200 0 1 3 0.65 Δx Ex. 9 Com. DMEM 40 2500 1 0.6 0.60 Δ x Ex. 10 Com. DMEM 40 0 1 0.60.60 x x Ex. 11 Com. DMEM 200 0 0.5 6 0.55 x x Ex. 12 Com. DMEM 200 00.75 4 0.53 x x Ex. 13 Com. DMEM 200 2500 1 3 0.47 x x Ex. 14 Com. DMEM200 0 0.75 4 0.37 x x Ex. 15 Com. DMEM 40 2500 1 0.6 0.16 Δ x Ex. 16

From the results of Table 2, it was found that the stirring state andthe non-stirring state of the second liquid show no difference. As isclear from Comparative Examples 1-16, even when the same through-hole isused, a smaller flow rate of the first liquid results in lower flowvelocity, and dispersion of fine structure is not achieved. From theabove, it was found that a medium composition in which a structure ispreferably dispersed can be obtained by simple injection according tothe production method of the present invention.

Experimental Example 2: Test Relating to Bore Diameter of Through-Holeof Nozzle Part

In Experimental Example 2, the same amount of the first liquid isinjected by the same pressing force from a syringe with the samespecifications, and changes in the flow rate and flow velocity, and thedispersed state (suspending property) of the beads and the state of thestructure (precipitate) as in the above-mentioned Experimental Example 1when the bore diameter of the through-hole of the nozzle part wasstepwisely changed were evaluated.

The first liquid, syringe, the second liquid, and conical tube used werethe same as those in the above-mentioned Experimental Example 1. InExperimental Example 2, to stepwisely change the bore diameter of thethrough-hole of the nozzle part, injection needles of various gauges(inner diameter) were mounted on the syringe tip. That is, in thisExperimental Example, the inner passage of the injection needle is thethrough-hole. The criteria of the evaluation of the dispersed state(suspending property) of the beads and the state of the structure(precipitate) were the same as those in the above-mentioned ExperimentalExample 1.

In Experimental Example 2, an injection needle with gauge number 18 wasused in Example 10, and the same gauge number and longer addition time(smaller flow rate) was used in Comparative Example 17.

Similarly, an injection needle with gauge number 20 was used in Example11, and the same gauge number and longer addition time (smaller flowrate) was used in Comparative Examples 18, 19.

Similarly, an injection needle with gauge number 22 was used in Example12, and the same gauge number and longer addition time (smaller flowrate) was used in Comparative Examples 20, 21.

Furthermore, as a reference test, an injection needle with a small borediameter gauge number 25 was used and tests by changing the additiontime were carried out as Comparative Examples 21, 22, 23.

The dispersed state (suspending action) of beads and the state ofstructure in each Experimental Example (Example, Comparative Example) isshown in Table 3.

TABLE 3 inner cross diameter of section of through- through- injecttionamount flow suspended hole hole time added flow rate velocity state ofnote relating to gauge [mm] [mm²] [second] [mm³] [mm/sec] [mm/sec] beadsprecipitate precipitate Ex. 10 18 0.94 0.69 0.2 800 4000 5767 ∘ ∘ noprecipitation Com. 18 0.94 0.69 1.4 800 571 824 x x large amount of Ex.17 precipitate Ex. 11 20 0.66 0.34 0.2 800 4000 11698 ∘ ∘ noprecipitation Com. 20 0.66 0.34 0.6 800 1333 3899 Δ Δ fibrousprecipitate Ex. 18 Com. 20 0.66 0.34 1.5 800 533 1560 x x large amountof Ex. 19 precipitate Ex. 12 22 0.48 0.18 0.3 800 2667 14744 ∘ ∘ noprecipitation Com. 22 0.48 0.18 0.6 800 1333 7372 ∘ Δ precipitated Ex.20 Com. 22 0.48 0.18 1.7 800 471 2602 x x large amount of Ex. 21precipitate Com. 25 0.32 0.08 1.6 800 500 6220 ∘ Δ gel-like suspendingEx. 22 matter Com. 25 0.32 0.08 2.5 800 320 3981 ∘ Δ gel-like suspendingEx. 23 matter Com. 25 0.32 0.08 3.3 800 242 3016 Δ x many precipitatesEx. 24 produced

In the following Table 4, the order of description in theabove-mentioned Table 3 was changed, and described separately inExamples 10-12 and Comparative Examples 17-24.

TABLE 4 inner cross diameter of section of through- through- injectionamount flow suspended hole hole time added flow rate velocity state ofnote relating to gauge [mm] [mm²] [second] [mm³] [mm³/sec] [mm/sec]beads precipitate precipitate Ex. 10 18 0.94 0.69 0.2 800 4000 5767 ∘ ∘no precipitation Ex. 11 20 0.66 0.34 0.2 800 4000 11698 ∘ ∘ noprecipitation Ex. 12 22 0.48 0.18 0.3 800 2667 14744 ∘ ∘ noprecipitation Com. 18 0.94 0.69 1.4 800 571 824 x x large amount of Ex.17 precipitate Com. 20 0.66 0.34 0.6 800 1333 3899 Δ Δ fibrous Ex. 18precipitate Com. 20 0.66 0.34 1.5 800 533 1560 x x large amount of Ex.19 precipitate Com. 22 0.48 0.18 0.6 800 1333 7372 ∘ Δ precipitated Ex.20 Com. 22 0.48 0.18 1.7 800 471 2602 x x large amount of Ex. 21precipitate Com. 25 0.32 0.08 1.6 800 500 6220 ∘ Δ gel-like Ex. 22suspending matter Com. 25 0.32 0.08 2.5 800 320 3981 ∘ Δ gel-like Ex. 23suspending matter Com. 25 0.32 0.08 3.3 800 242 3016 Δ x manyprecipitates Ex. 24 produced

As is clear from the results of Table 4, even when the inner diameter ofthe through-hole is the same, good bead dispersed state (good suspendingproperty) and good structure state (no precipitation) cannot besatisfied simultaneously unless the first liquid is injected at anappropriate flow rate.

As is also clear from the results of Comparative Examples 17-24, evenwhen the cross section area of the through-hole is within the range ofthe present invention, good bead dispersed state (good suspendingproperty) and good structure state (no precipitation) cannot besatisfied simultaneously when the first liquid is added to the secondliquid at a flow rate of not more than 1.7 mL/sec.

Experimental Example 3: Test Relating to Inject Direction of the FirstLiquid

In Experimental Example 2, in injecting the first liquid from thesyringe into the container, whether the dispersed state of the beads(suspending property) varies between an injection direction of from thetop to the lower side, and an injection direction of from the bottom tothe upper side by setting the container upside down, was evaluated.

The first liquid, syringe, second liquid, and conical tube used were thesame as those in the above-mentioned Experimental Example 1.

The dispersed state (suspending action) of beads in each ExperimentalExample is shown in Table 5.

TABLE 5 DAG second liquid amount of final temperature amount firstliquid concentration container, syringe suspending No. kind [° C.] [mL][mL] [wt %] orientation when mixing action note a1 DEEM-h 4 40 0.8 0.01550 mL tube, 1 mL syringe ∘ good injected from top a2 DEEM-h 4 40 0.80.015 50 mL tube, 2.5 mL syringe ∘ good injected from bottom a3 DEEM-h 440 0.8 0.015 50 mL tube, 1 mL syringe ∘ good injected from bottom a4DEEM-h 4 50 1.0 0.015 50 mL tube, 1 mL syringe ∘ many fine injected fromtop bubbles a5 DEEM-h 4 50 1.0 0.015 50 mL tube, 2.5 mL syringe ∘ manyfine injected from top bubbles a6 DEEM-h 4 50 1.0 0.015 50 mL tube, 1 mLsyringe ∘ good injected from bottom a7 DEEM-h 4 40 0.8 0.015 50 mL tube,2.5 mL syringe ∘ many fine injected from top bubbles a8 DEEM-h 4 50 1.00.015 50 mL tube, 2.5 mL syringe ∘ good injected from bottom

As is clear from the results of Table 5, it was found that theproduction method of the present invention affords a preferablesuspending action irrespective of the injection direction.

Experimental Example 4: Test Relating to Shape of Container, Distancefrom Outlet Opening of Nozzle Part to Liquid Surface

In Experimental Example 4, an influence of the ratio of container length(size in depth direction) L and outer diameter D of container (aspectratio L/D), the ratio of the second liquid depth Z maintained in thecontainer and inner diameter d of container (aspect ratio Z/d), and thedistance from outlet opening of the nozzle part to liquid surface on thesuspending property during mixing was examined.

The dispersed state (suspending action) of beads in each ExperimentalExample is shown in Table 6.

TABLE 6 outer dimensions of distance inner size of container body fromliquid outer DAG DAG discharge immersion part capac- diam- liquid liquidopening to inner ity length eter concen- amount stir- liquid depth diam-second liquid type L D tration added addition ring surface Z eter dfloat- No. kind amount (mL) (mm) (mm) L/D (wt %) (mm³) method (rpm) (mm)(mm) (mm) Z/d ability note b1 DMEM 5000 15 118 16 7.4 1 75 syringe +2600 73 43 14 3.1 ∘ high LD (HG) tip container needle b2 DMEM 10000 50115 28 4.1 0.75 200 container 0 85 28 26 1.1 x distance (HG) with toliquid nozzle surface x part b3 DMEM 20000 50 115 28 4.1 0.75 400container 0 67 46 26 1.8 ∘ distance (HG) with to liquid nozzle surface ∘part b4 DMEM 30000 50 115 28 4.1 0.75 600 container 0 48 65 26 2.5 ∘distance (HG) with to liquid nozzle surface ∘ part b5 DMEM 40000 50 11528 4.1 0.75 800 container 0 30 83 26 3.2 ∘ distance (HG) with to liquidnozzle surface ∘ part b6 DMEM 50000 50 115 28 4.1 0.75 1000 container 012 101 26 3.9 ∘ distance (HG) with to liquid nozzle surface ∘ part b7DMEM 140000 225 138 60 2.3 1 2100 container 2500 64 72 58 1.2 x low LD(HG) with container nozzle and liquid part surface distance b8 DMEM160000 225 138 60 2.3 1 2400 container 2500 56 80 58 1.4 ∘ low LD (HG)with container nozzle and liquid part surface distance b9 DMEM 180000225 138 60 2.3 1 2700 container 2500 49 87 58 1.5 ∘ low LD (HG) withcontainer nozzle and liquid part surface distance b10 DMEM 200000 225138 60 2.3 1 3000 container 2500 41 95 58 1.6 ∘ low LD (HG) withcontainer nozzle and liquid part surface distance

As is clear from the results of Table 6, even when a container with thesame aspect ratio L/D is used, when the immersion part is shallow andthe aspect ratio Z/d is small, and when the distance from the outletopening of the nozzle part to the liquid surface is large, preferablesuspending property cannot be achieved. The results show that, under theconditions of the above-mentioned Experimental Example 4, preferablesuspending property cannot be achieved when the aspect ratio Z/d of theimmersion part is not more than 1.24, and preferable suspending propertycan be obtained when the aspect ratio Z/d exceeds 1.24.

Experimental Example 5: Test Relating to Suspending Property when Kindof Medium is Changed

In Experimental Example 5, suspending property of a liquid mediumcomposition produced by changing the kind of the medium as the secondliquid was examined. The temperature of the second liquid contained inthe container was maintained at 37° C., and the temperature of the firstliquid was set to room temperature (RT).

The dispersed state (suspending action) of beads and the state ofstructure in each Experimental Example is shown in Table 7.

TABLE 7 second liquid B first liquid A evaluation results amount amountsuspended added stirring temperature concentration added temperaturestate of state of test No kind [mL] [rpm] [° C.] [%] [mL] [° C.] beadsstructure c1 RPMI 30 0 37 0.8 0.8 RT ∘ ∘ c2 DMEM 40 0 37 0.8 0.8 RT ∘ ∘c3 DMEM-F12 40 0 37 0.8 0.8 RT ∘ ∘ c4 Ham's 40 0 37 0.8 0.8 RT ∘ ∘ F12c5 EMEM 40 0 37 0.8 0.8 RT ∘ ∘

As is clear from the results of Table 7, even when the second liquid isdifferent, the first liquid and the second liquid can be mixed well, andpreferable suspending property can be obtained.

Experimental Example 6: Cell Proliferation Experiment of A549 CellsUsing Low Adhesion Plate

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.)was suspended in ultra pure water (Milli-Q water) to 0.3% (w/v) or 0.75%(w/v), dissolved by stirring with heating at 90° C., and this aqueoussolution was autoclave sterilized at 121° C. for 20 min. Using 0.3%solution, a medium composition was prepared by adding deacylated gellangum (final concentration 0.015% (w/v)) to DMEM medium (manufactured byWAKO) containing 10% (v/v) fetal bovine serum. Fetal bovine serum wasadded at 10% (v/v) to a medium composition prepared by a conventionalmethod (method using homomixer as described in Example of JP-B-5629893)or Example 7.

Human lung cancer cell line A549 (manufactured by DS PHARMA BIOMEDICAL)was seeded in the above-mentioned medium composition containing fetalbovine serum and added with deacylated gellan gum (conventional method)or the medium composition of Example 7 at 33333 cells/mL, and dispensedto the wells of a 96 well flat bottom ultra-low adhesive surfacemicroplate (Corning Incorporated manufactured by, #3474) at 150 μL per 1well. As a negative control, A549 cells were suspended in theabove-mentioned medium free of deacylated gellan gum and dispensed.Successively, the plate was cultured in a static state in a CO₂incubator (37° C., 5% CO₂) for 7 days. ATP reagent (150 μL,CellTiter-Glo™ Luminescent Cell Viability Assay, manufactured byPromega) was added to the culture medium on day 0 immediately afterseeding and 7 days after culture and suspended therein, stood for about10 min at room temperature, and the luminescence intensity (RLU value)was measured by FlexStation3 (manufactured by Molecular Devices), fromwhich the luminescence with the medium alone was subtracted to measurethe number of viable cells. The above experiment was performed 3 times.

A method using the medium composition by the production method of thepresent invention showed the same level of proliferation of A549 cellsas compared to the conventional method. RLU values (ATP measurement,luminescence intensity) at culture day number 0 and 7 days later ofstatic culture are shown in Table 8 as a mean of 3 tests.

TABLE 8 culture day number 0 7 negative control 9361 29610 conventionalmethod deacylated gellan gum 7811 97182 production method of deacylatedgellan gum 7500 95840 present invention

As is clear from the results of Table 8, the medium composition producedby the production method of the present invention was suggested toenable suspension culture of cells and promote cell proliferation, aswith the medium composition produced by the conventional method.

Experimental Example 7: A549 Cell Proliferation Suppressive Test UsingTrametinib and MK-2206 in Low Adhesion Plate

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.)was suspended in ultra pure water (Milli-Q water) to 0.3% (w/v) or 0.75%(w/v), dissolved by stirring with heating at 90° C., and this aqueoussolution was autoclave sterilized at 121° C. for 20 min. Using 0.3%solution, a medium composition was prepared by adding deacylated gellangum (final concentration 0.015% (w/v)) to DMEM medium (manufactured byWAKO) containing 10% (v/v) fetal bovine serum. Fetal bovine serum wasadded at 10% (v/v) to a medium composition prepared in Example 7.

Human lung cancer cell line A549 (manufactured by DS PHARMA BIOMEDICAL)was seeded in the above-mentioned medium composition added withdeacylated gellan gum (conventional method) or the medium composition ofExample 7 at 14800 cells/mL, and dispensed to the wells of a 96 wellflat bottom ultra-low adhesive surface microplate (Corning Incorporatedmanufactured by, #3474) at 135 μL per 1 well. As a negative control,A549 cells were suspended in the above-mentioned medium free ofdeacylated gellan gum and dispensed. Each plate was cultured in a staticstate in a CO₂ incubator (37° C., 5% CO₂). On day 1 of culture, mediumcompositions (conventional method and the production method of thepresent invention) containing each anticancer agent at 10-foldconcentration and deacylated gellan gum (final concentration 0.015%(w/v)), and a medium composition containing each anticancer agent aloneat 10-fold concentration (non-addition method), 15 μL each, was added toa final concentration of 0.001 to 30 μM, and culture was continued for 7days. As the anticancer agent, Trametinib (manufactured by Santa Cruz,MEK inhibitor) and MK-2206 (manufactured by Santa Cruz, Akt inhibitor)were used. ATP reagent (150 μL, CellTiter-Glo™ Luminescent CellViability Assay, manufactured by Promega) was added to the culture mediaon days 5 and 8 and suspended therein, stood for about 10 min at roomtemperature, and the luminescence intensity (RLU value) was measured byFlexStation3 (manufactured by Molecular Devices), from which theluminescence with the medium alone was subtracted to measure the numberof viable cells.

The suppressive effect of each anticancer agent on A549 cellproliferation was not different and similar between the mediumcomposition produced by the production method of the present inventionand the medium composition produced by a conventional method, and it wasfound that the medium composition of the present invention also showsstrong efficacy of MK-2206. In addition, % control of RLU value (ATPmeasurement, luminescence intensity) on day 4 of static culture is shownin Table 9, and % control of RLU value (ATP measurement, luminescenceintensity) on day 7 of static culture is shown in Table 10.

TABLE 9 non- conven- production method addition tional of presentculture conditions method method invention % Control DMSO 100 100 100Paclitaxel 0.001 μM 112 98 91 Paclitaxel 0.01 μM 57 31 32 Trametinib0.001 μM 76 85 82 Trametinib 0.003 μM 57 65 60 Trametinib 0.01 μM 65 6157 Trametinib 0.03 μM 46 40 41 MK-2206 0.03 μM 108 100 85 MK-2206 0.1 μM99 91 74 MK-2206 0.3 μM 93 71 60 MK-2206 1 μM 78 50 43 MK-2206 3 μM 6238 33

TABLE 10 non- conven- production method addition tional of presentculture conditions method method invention % Control DMSO 100 100 100Paclitaxel 0.001 μM 116 90 84 Paclitaxel 0.01 μM 49 12 12 Trametinib0.001 μM 76 81 76 Trametinib 0.003 μM 48 52 48 Trametinib 0.01 μM 56 4845 Trametinib 0.03 μM 39 25 24 MK-2206 0.03 μM 124 92 80 MK-2206 0.1 μM101 83 69 MK-2206 0.3 μM 103 63 54 MK-2206 1 μM 85 38 34 MK-2206 3 μM 7626 21

As is clear from the results of Table 9, Table 10, the mediumcomposition produced by the production method of the present inventionwas suggested to enable suspension culture of cells and potentiate aproliferation-suppressive effect of the anticancer agent against cancercells, as with the medium composition produced by the conventionalmethod.

Experimental Example 8: Comparison with Monolayer Culture Method inProliferation Action on Panc02, 03 Cell Stimulated by Each Growth Factor

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.)was suspended in ultra pure water (Milli-Q water) to 0.75% (w/v),dissolved by stirring with heating at 90° C., and this aqueous solutionwas autoclave sterilized at 121° C. for 20 min. In the same manner as inExample 7, a medium composition was produced by adding deacylated gellangum (final concentration 0.02% (w/v)) to RPMI1640 medium (manufacturedby WAKO) containing fetal bovine serum at 15% (v/v) and used as Example13.

Human pancreatic cancer cell line Panc02, 03 (manufactured by ATCC) wasseeded at 37000 cells/mL in the medium composition of Example 13(manufactured by the production method of the present invention), anddispensed to the wells of a 96 well flat bottom ultra-low adhesivesurface microplate (Corning Incorporated manufactured by, #3474) at 135μL per 1 well. As a negative control, Panc02, 03 cells were suspended inthe above-mentioned medium free of deacylated gellan gum and dispensed.As a monolayer culture method, human pancreatic cancer cell line Panc02,03 cells were seeded at 2200 cells/mL in the above-mentioned mediumcomposition free of deacylated gellan gum, and dispensed to the wells ofa 96 well flat bottom microplate (manufactured by Corning Incorporated,#3585) at 135 μL per 1 well. Each plate was cultured in a static statein a CO₂ incubator (37° C., 5% CO₂). On day 1 of culture, mediumcompositions (manufactured by conventional method and the productionmethod of the present invention) containing 10-fold concentration ofhuman HB-EGF (manufactured by PEPROTECH) to a final concentration of 30,100 ng/mL, 10-fold concentration of human EGF (manufactured byPEPROTECH) to 3, 30 ng/mL, 10-fold concentration of human FGF2(manufactured by PEPROTECH) to 10, 100 ng/mL, 10-fold concentration ofhuman TGF-β1 (manufactured by PEPROTECH) to 3, 30 ng/mL, 10-foldconcentration of human PDGF-BB (manufactured by PEPROTECH) to 10 ng/mLor 10-fold concentration of human IGF-1 (manufactured by PEPROTECH) to10, 100 ng/mL, and a final concentration 0.015% (w/v) of deacylatedgellan gum, each 15 μL, were added. In a monolayer culture group and anegative control group, a medium composition containing a 10-foldconcentration of each growth factor alone was added by 15 μL each.Culturing was continued for 5 days. ATP reagent (150 μL, CellTiter-Glo™Luminescent Cell Viability Assay, manufactured by Promega) was added tothe culture media on day 6 and suspended therein, stood for about 10 minat room temperature, and the luminescence intensity (RLU value) wasmeasured by FlexStation3 (manufactured by Molecular Devices), from whichthe luminescence with the medium alone was subtracted to measure thenumber of viable cells.

As a result, it was found that the efficacy of human HB-EGF and humanEGF is strongly shown by Panc02, 03 cell proliferation test method usingthe medium composition by the production method of the presentinvention, as compared to a monolayer culture method and negativecontrol group. In addition, % control of RLU value (ATP measurement,luminescence intensity) on day 6 of static culture is shown in Table 11.

TABLE 11 monolayer negative culture conditions culture control Exampleon day 8 group group 13 % Control no addition 100 100 100 human HB-EGF30 ng/mL 99 132 118 human HB-EGF 100 ng/mL 98 134 202 human EGF 3 ng/mL105 117 108 human EGF 30 ng/mL 97 136 187 human FGF-2 10 ng/mL 108 118107 human FGF-2 100 ng/mL 102 99 100 human TGF-β1 3 ng/mL 95 114 102human TGF-β1 30 ng/mL 99 122 106 human IGF-1 10 ng/mL 101 119 100 humanIGF-1 100 ng/mL 98 121 110 human PDGF-BB 10 ng/mL 98 128 101

As is clear from the results of Table 11, it was suggested that a mediumcomposition produced by the production method of the present inventionenables suspension culture of cells and promotes cell proliferation byHB-EGF or EGF stimulation, as with a medium composition produced by aconventional method.

INDUSTRIAL APPLICABILITY

According to the present invention, any liquid (particularly liquidmedium) containing a linking substance such as divalent metal cation andthe like can be easily mixed with a liquid containing a particularcompound, and a liquid medium composition containing a fine structuredispersed therein can be produced.

This application is based on patent application No. 2015-078795 filed inJapan (filing date: Apr. 7, 2015), the contents of which areincorporated in full herein.

1. A kit for carrying out a production method of liquid mediumcomposition, which kit comprises at least a first container, a syringe,and a second container, wherein the first container contains a firstliquid comprising a particular compound of a particular compound whichis a polymer compound having an anionic functional group, and capable offorming a structure by linking via a divalent metal cation, whichstructure being capable of suspending a cell or a tissue, the syringefunctions as a supply device for feeding out the first liquid, and thesecond container contains a second liquid comprising a linking substancewhich is a divalent metal cation, and the second container comprises abody and a lid, wherein said body or lid is provided with a nozzle parthaving a through-hole communicating the outside of the container and theinside of the container, said through-hole has a cross-sectional area of0.01 mm²-5.00 mm², the nozzle part is provided on the lid of thecontainer, a tubular component for fitting a syringe tip protrudes froman outer surface of the lid at a container external side of the nozzlepar.
 2. The kit according to claim 1, wherein the syringe is furtherequipped with a tubular component to be inserted in the first containerand used to suck the first liquid, said tubular component comprising athin-tube part having an outer diameter permitting insertion into thefirst container, and a length enabling suction of the first liquid fromthe first container, and a connecting part at one end of the thin-tubepart, mountable on the tip of the cylinder of the syringe.
 3. The kitaccording to claim 1, wherein the second container is further equippedwith a sealing lid free of a nozzle part and configured to be able toseal the inside of the body of the container, and the lid of the secondcontainer and the sealing lid can be compatibly mounted on an openingpart of the body of the container.
 4. The kit according to claim 3,wherein the syringe is further equipped with a tubular component to beinserted in the first container and used to suck the first liquid, saidtubular component comprising a thin-tube part having an outer diameterpermitting insertion into the first container, and a length enablingsuction of the first liquid from the first container, and a connectingpart at one end of the thin-tube part, mountable on the tip of thecylinder of the syringe.